The present invention relates to a method of preparing highly purified, stroma-free, non-hepatitic human and animal hemoglobin solutions.
Hemoglobin solutions have the capacity to transport oxygen in vivo independent of erythrocytes. For the compatibility of such solutions, however, it is necessary to remove as completely as possible the plasma and cell components (plasma proteins, stroma, etc.) of the blood from which the hemoglobin is to be released by hemolysis.
Various methods of preparing hemoglobin solutions are in use.
The first stage of all these methods consists of washing the erythrocytes to remove the plasma proteins. In most of the methods known up to now the erythrocytes are treated with isotonic electrolyte solutions and separated by centrifugation as described for example in German Pat. No. 2 248 475.
U.S. Pat. No. 4,439,357 describes a method that is also appropriate for large-scale preparation and involves sedimentation for the purpose of washing the erythrocytes.
The next step in all preparation methods is hemolysis of the erythrocytes followed by separating out of the stroma components, which can be done by filtration, centrifugation, recrystallization of the hemoglobin, or by a combination of these procedures (U.S. Pat. No. 4,439,357; De Venuto et al., "Appraisal of Hemoglobin Solution as a Blood Substitute, Surgery, Gynecology & Obstetrics, 149 [September 1979], 417-36; De Venute et al., "Characteristics of Stroma-free Hemoglobin Prepared by Crystallization," J. Clin. Med, March 1977, 509-16; Feola et al., "Development of a Bovine Stroma-free Hemoglobin Solution as a Blood Substitute," Surgery, Gynecology & Obstetrics, November 1983).
This maximally possible removal of the stroma is especially significant because residual stroma lipids can possibly affect blood clotting and provoke renal damage.
All of the methods of preparing hemoglobin solutions employed up to the present time, however, exhibit decisive drawbacks. Present procedures for washing the erythrocytes have led to products that still contain plasma proteins. Complete removal of the plasma proteins by repeated washing of the erythrocytes or recrystallization of the hemoglobin results in considerable losses of yield and is uneconomical on a large scale.
For this reason it has also been impossible by conventional methods to prepare hemoglobin solutions from animal blood for possible utilization in human medicine because the residual proteins in the solutions can lead to immune reactions when employed with humans.